Radiometal-binding peptide analogues

ABSTRACT

Novel metal binding ligands are disclosed that may be coupled to peptides for use in methods of diagnosis and therapy. Peptides containing the ligands are produced using a method wherein ligand introduction or cyclization can be conducted at any point during the synthesis of the peptide. Such peptide derivatives are readily labeled with radiometals, such as isotopes of rhenium or technetium, while retaining their ability to tightly bind specific peptide receptors.

This application is a division of application Ser. No. 08/893,749, filedJul. 11, 1997, now U.S. Pat. No. 6,126,916, which claims the benefit ofProvisional application Ser. No. 60/021,662, filed Jul. 2, 1996.

BACKGROUND OF THE INVENTION

This invention provides derivatives of biologically useful cyclic andacyclic peptides in which one or more amino acid side chains or asegment attached to the peptide chain contain chelating moieties thatcan tightly bind metal ions, including radionuclides. The labeledpeptides carry the metal to specific in vivo targets such as receptorsand antigens, and are useful for radiodiagnostic imaging, therapy andradiotherapy. New methods for preparing the peptides are also provided.

Radiolabeled peptides are useful in the diagnosis and therapy of avariety of human disease states that are characterized by overexpressionof peptide hormone receptors. Thus, for example, it has been shown thatradiolabeled analogues of LHRH (luteinizing hormone releasing hormone)and somatostatin selectively bind to hormone-sensitive tumorscharacterized by cell-surface overexpression of LHRH hormone receptors.Similarly, peptide hormone analogues such as ¹²³I-vasoactive intestinalpeptide (VIP), ^(99m)Tc-P829, ¹¹¹In-DTPA Octreotide and¹¹¹In-bisMSH-DTPA have been used to image human tumors that over expressVIP, somatostatin, somatostatin and melanocyte stimulating hormone (MSH)receptors respectively. See: Virgolini et al. Engl. J. Med. 169:1116(1994); Virgolini et al. J. Nucl. Med. 36:1732, (1995); Lister-James etal. Nucl. Med., 36, 91P, #370, 1995 meeting abstract; Pearson et al. J.Med. Chem. 39:1361, (1996); Krenning et al. J. Nucl. Med., 33:652(1992); and Wraight et al. Brit. J. Radiol. 65:112 (1992).

Many tyrosine-containing peptides may be labeled with ¹²⁵I by well knownmethods and used for receptor binding studies. For example, theincidence of VIP receptor upregulation has been studied in vitro in awide range of cancer types using ¹²⁵I-[Tyr¹⁰]-VIP as the radioligand.See Reubi, Nucl. Med. 36:1846 (1995). The VIP receptor was detected in awide variety of cancer types, including breast, prostate, ovarian,pancreatic, endometrial, bladder, colon, esophageal, SCLC, astrocytoma,glioblastoma, meningioma, pheochromocytoma, lymphoma, neuroblastomaadenoma, and GEP tumors. An iodinated VIP analogue ¹²³I-[Tyr¹⁰]-VIP hasalso been used to image VIP receptor-rich tumors in humans. SeeVirgolini et al, supra.

The use of radioiodine for in vivo diagnostic and therapeutic uses hasdistinct disadvantages, however. ¹²³I, the most useful isotope in vivo,is very expensive ($45.30/mCi) and must be produced in a cyclotron. Thisisotope, furthermore, has a half-life of only 13.2 hours, therebyrequiring that it be produced in a geographic location close to whereany radioiodinated imaging agent must be used. Other radioisotopes, suchas ^(99m)Tc and ¹⁸⁸Re are preferred for diagnostic and therapeutic uses,respectively. ^(99m)Tc, for example, is inexpensive ($0.50/mCi), isreadily available (produced in a generator from ⁹⁹Mo, a reactorproduct), and has an ideal gamma emission energy for imaging with agamma camera.

Some peptides either directly contain, or are amenable to theintroduction of, residues that allow direct binding of radiometals suchas ^(99m)Tc and ¹⁸⁸Re to the peptide. For example, somatostatin containsa disulfide bond that, upon reduction, provides twosulfhydryl-containing cysteine side chains that can directly bind^(99m)Tc. See U.S. Pat. No. 5,225,180. See also WO 94/28942, WO 93/21962and WO 94/23758. Complexes of this type tend, however, to beheterogeneous and unstable, which limits their clinical utility.Moreover, the use of free sulfhydryls in this manner limits theradiometals which can be used to label the peptide to those that tightlybind free S—H groups. This method further suffers from the problem thatdirect binding of the metal to an amino acid side chain can greatlyinfluence the peptide conformation, thereby deleteriously altering thereceptor binding properties of the compound.

Most peptides either do not contain a metal-binding amino acid sequencemotif or, for various reasons such as those described supra, are notamenable to suitable sequence modifications that would permitintroduction of such a motif. Some means of rendering the peptidecapable of binding radiometals must therefore be introduced into thepeptide. A preferred approach is to attach a metal binding ligand to aspecified site within the peptide so that a single defined, stable,complex is formed. The ligands used to bind metals often contain avariety of heteroatoms such as nitrogen, sulfur, phosphorous, and oxygenthat have a high affinity for metals.

Chelates have conventionally been attached via covalent linkages to theN-terminus of a peptide or peptide analogue, following independentsynthesis of the peptide and chelate moieties. For example, Maina et al.have described the coupling of a tetra-amine chelator to the N-terminusof a somatostatin analogue, allowing ^(99m)Tc labeling of the peptide.See J. Nucl. Biol. Med. 38:452 (1994). Coupling in this manner is,however, undesirable when the N-terminus of the peptide plays animportant role in its receptor binding properties. Accordingly,application of this method is limited by the requirement that theN-terminus of the peptide accommodate the presence of a (usuallysterically bulky) chelator without deleteriously affecting the bindingproperties of the peptide.

Alternatively, chelating agents have been introduced into peptide sidechains by means of, site-selective reactions involving particular aminoacid residues. For example, the lysine residue at position 6 of LHRH hasbeen directly acylated with a chelating group. See Bajusz, S. et al.Proc. Natl. Acad. Sci. USA 86:6313 (1989). This method is inherentlylimited by the lack of chemical selectivity available when more than oneside chain can potentially react with the chelator, or when the peptidesequence does not contain an amino acid that can be derivatized in thisway. A further limitation of this approach can arise when multidentateligands are used. A single ligand molecule can react with multiplepeptide molecules resulting in the formation of significant amounts ofcross-linked products.

Chelating agents have been introduced on the side chain of a peptidethrough tris amino-acids as described by Dunn T. J. et al. WO 94/26294.This method does not provide a method for cyclizing the peptides. Theside chain protecting groups used to introduce the ligand described inthis work are the same as those typically used for peptide amidecyclization. See Felix et al. Int. J. Peptide Protein Res. 32:441(1988).

A fully-protected BAT (bisaminothiol) chelating agent has beensynthesized and coupled to the side chain of a lysine residue, whichcould then be incorporated into a peptide. See Dean et al. WO 93/25244.These fully protected precursors are very time consuming, expensive andcumbersome to prepare. The difficulty and expense of preparing suchprecursors make this method untenable for preparing a diverse array ofligands attached to the variety of linkers that is needed to design ametal carrying targeting agent.

One potential solution to this problem is to use a protecting groupstrategy that allows selective coupling of a chelator moiety tospecified positions within a peptide chain. The diversity of chemicalreactivities present within the amino acid side chains of a peptide has,however, led to difficulties in achieving sufficient selectivity insite-specific deprotection of protecting groups. This lack ofselectivity has also heretofore hampered efforts to selectivelydeprotect two or more different functional groups within a peptide toallow coupling of these groups in, for example, a cyclic peptide.

Edwards et al. J. Med. Chem. 37:3749 (1994) have disclosed a fragmentmethod of assembling a cyclic disulfide on a resin with a subsequentattachment of an intact ligand (DTPA). This approach afforded the knownsomatostatin targeting agent DTPA-Octreotide. This approach wasspecifically designed for the preparation of a known compound. A moretypical situation, however, requires that a variety of labeled peptidesto optimize binding to a particular target. Such a situation requires,therefore, a broader approach allowing the assembly of multiple ligands,best assembled in fragments, placed at any point desired in a sequencewhich can also be cyclized at a variety of positions in the peptidesequence.

Additional considerations for the synthesis of peptides that canselectively bind metals include the effect of the chelate on theconformation of the peptide. Most peptides are highly conformationallyflexible, whereas efficient receptor binding usually requires that apeptide adopt a specific conformation. Whether or not the peptide canadopt this specific conformation is greatly influenced by charge andhydrophilic/hydrophobic interactions, including the effects of acovalently attached metal chelating moiety. It is possible to enhancepeptide receptor affinity and selectivity by restricting theconformations that the peptide can adopt, preferably locking the peptideinto an active conformation. This is often most readily achieved bypreparing cyclic peptides. Cyclic peptides have the added advantage ofenhanced resistance to proteases, and therefore frequently demonstrate alonger biological half-life than a corresponding linear peptide.

Peptides can be cyclized by a variety of methods such as formation ofdisulfides, sulfides and, especially, lactam formation between carboxyland amino functions of the N- and C-termini or amino acid side chains.However, the plethora of functionality within a peptide chain typicallymeans that, for all but the shortest peptides, selective couplingbetween two desired functional groups within a peptide is very difficultto achieve.

It is apparent, therefore, that cyclic peptides that can chelate metalsions while retaining the ability to specifically bind with high affinityto a receptor are greatly to be desired. It is also desirable to have ameans of attaching a chelating moiety to any predetermined positionwithin a peptide, and to have a means of selectively forming cyclicpeptides between any two preselected positions within a peptide chain.Additionally, it is desirable to have access to a method that wouldallow a chelating moiety to be coupled to a peptide at any desired stageduring peptide synthesis.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide peptidesthat can bind radionuclides while retaining the ability to specificallybind to the peptide receptor. It is a further object of the invention toprovide methods of preparing and radiolabeling peptides that can bindradionuclides while retaining the ability to specifically bind to thepeptide receptor. It is a still further object of the invention toprovide diagnostic and therapeutic methods of using the radiolabeledpeptides to image or treat a tumor, an infectious lesion, a myocardialinfarction, a clot, atherosclerotic plaque, or a normal organ or tissue.

In accomplishing the foregoing objects of the invention, there has beenprovided, in accordance with one aspect of the current invention, apeptide comprising a radiometal-binding moiety, wherein said bindingmoiety comprises the structure I:

where R¹, R², and R³ independently are selected from the groupconsisting of H, lower alkyl, substituted lower alkyl, cycloalkyl,substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkaryl, and a protecting group thatcan be removed under the conditions of peptide synthesis, provided thatat least one of R¹, R², or R³ is H. R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰independently are selected from the group consisting of H, lower alkyl,substituted lower alkyl, aryl, and substituted aryl, or R⁴ and R⁶together optionally form a direct bond. R⁸ and R⁹ together or R⁷ and R⁹together may form a cycloalkyl or substituted cycloalkyl ring, and NR¹⁰is located at the N-terminus of said peptide, or is located on an aminoacid side chain of said peptide.

In preferred embodiments of the invention, R¹ is H, R³ is H, R⁴ is H, orR⁴ and R⁶ together form a direct bond. In other preferred embodiments,R² is lower alkyl or substituted or unsubstituted phenyl, or morepreferably methyl or phenyl. In other preferred embodiments, R⁸ and R⁹are methyl.

In accordance with another aspect of the invention, the peptides furthercomprise a bound metal atom. In preferred embodiments the metal atom is^(99m)Tc, ¹⁸⁶Re, or ¹⁸⁸Re.

In accordance with yet another aspect of the invention, there isprovided a method of preparing a metal-chelating composition, where asolution of a peptide comprising a radiometal-binding moiety iscontacted with stannous ions, where the binding moiety has the structureset forth above, followed by contacting the solution with aradionuclide, and recovering the radiolabeled peptide. In a preferredembodiment of the method the radionuclide is ¹⁸⁸Re- or ¹⁸⁶Re-perrhenateor ⁹⁹Tc-pertechnetate.

In accordance with still another aspect of the invention, there isprovided a method of imaging a tumor, an infectious lesion, a myocardialinfarction, a clot, atherosclerotic plaque, or a normal organ or tissue,comprising administering to a human patient a radiolabeled peptide,together with a pharmaceutically acceptable carrier, and, after asufficient time for said radiolabeled peptide to localize and fornon-target background to clear, the site or sites of accretion of saidradiolabeled peptide are detected by an external imaging camera, wherethe radiolabeled peptide is prepared by contacting a solution of apeptide with stannous ions, where the peptide comprises aradiometal-binding moiety having the structure set forth above, and thencontacting said solution with a radionuclide and recovering theradiolabeled peptide.

In accordance with another aspect of the invention there are providedpeptides having a structure selected from the group consisting of:

-   (Chel)γAbuNleDHF _(d) RWK-NH₂, (SEQ ID NO:1)-   (Chel)γAbuHSDAVFTDNYTRLRKQMAVKKYLNSILN-NH₂, (SEQ ID NO:2)-   KPRRPYTDNYTRLRK(Chel)QMAVKKYLNSILN-NH₂, (SEQ ID NO:3)-   (Chel)γAbuVFTDNYTRLRKQMAVKKYLNSILN-NH₂,    (Chel)γAbuYTRLRKQMAVKKYLNSILN-NH₂, (SEQ ID NO:4)-   HSDAVFTDNYTRLRK(Chel)QMAVKKYLNSILN-NH₂, (SEQ ID NO:5)-   (SEQ ID NO:6)<GHWSYK(Chel)LRPG-NH₂, <GHYSLK(Chel)WKPG-NH₂, (SEQ ID    NO:7)-   AcNal_(d) Cpa_(d) W_(d) SRK_(d) (Chel)LRPA_(d)-NH₂, (SEQ ID NO:8)-   (SEQ ID NO:9) (Chel)γAbuSYSNleDHF_(d) RWK-NH₂,    Ac-HSDAVFTENYTKLRK(Chel)QNleAAKKYLNDLKKGGT-NH₂, (SEQ ID NO:10)-   (SEQ ID NO:12) Nal_(d) Cpa_(d) W_(d) SRK_(d) (Chel)WKPG-NH₂,    <GHWSYK_(d)(Chel)LRPG-NH₂, (SEQ ID NO:13)-   (SEQ ID NO:14) AcK(Chel)F_(d) CFW _(d) KTCT-OH, AcK(Chel)DF_(d) CFW    _(d) KTCT-OH, (SEQ ID NO:15)-   (SEQ ID NO:14) AcK(Chel)F_(d) CFW _(d) KTCT-ol, AcK(Chel)DF_(d) CFW    _(d) KTCT-ol, (SEQ ID NO:15)-   (SEQ ID NO:16) (Chel)DF_(d) CFW _(d) KTCT-OH, K(Chel)DF_(d) CFW _(d)    KTCT-ol, (SEQ ID NO:15)-   (SEQ ID NO:17) K(Chel)KKF_(d) CFW _(d) KTCT-ol, K(Chel)KDF_(d) CFW    _(d) KTCT-OH, (SEQ ID NO:18)-   (SEQ ID NO:19) K(Chel)DSF_(d) CFW _(d) KTCT-OH, K(Chel)DF_(d) CFW    _(d) KTCT-OH, (SEQ ID NO:15)-   (SEQ ID NO:20) K(Chel)DF_(d) CFW _(d) KTCD-NH₂, K(Chel)DF_(d) CFW    _(d) KTCT-NH₂, (SEQ ID NO:15)-   (SEQ ID NO:18) K(Chel)KDF_(d) CFW _(d) KTCT-NHNH₂, AcK(Chel)F_(d)    CFW _(d) KTCT-NHNH₂, (SEQ ID NO:14)-   (SEQ ID NO:14) K(Chel)F_(d) CFW _(d) KTCT-ol, and F_(d) CFW _(d)    KTCTK(Chel)-NH₂, (SEQ ID NO:21)    wherein (Chel) is a radiometal-binding moiety having the structure    set forth above.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION

The present invention provides new chelating moieties that can becovalently linked to peptides, cyclic peptides and peptide analogues.The chelating moieties allow the peptides, cyclic peptides and peptideanalogues to stably bind metals, especially radiometals. Methods ofpreparing these chelators, peptides and peptide analogues are alsoprovided. The peptides and peptide analogues are prepared bysite-specifically introducing the metal-chelating moieties into peptidesthat are synthesized by solid-phase or solution phase methods. Thechelating moieties may be attached to an amine-bearing side-chain of anamino acid within the peptide chain, or may be attached to theN-terminus of the peptide. Peptides according to the invention include,but are not limited to, cyclic metal-binding analogues of LHRH,vasoactive intestinal peptide (VIP), heregulins (erbB binding peptides)α, β1, β2, and β3, melanotropin (α-MSH), somatostatin, calcitonin,epidermal growth factor, gonadotrophin releasing hormone, heregulinsgrowth hormone releasing hormone, dynorphin, calcitonin gene-relatedpeptide, vasotocin, mesotonin, adrenocorticotropic hormone,corticotropin, gonadotropin, prolactin, vasopressin, oxytocin, substanceP, substance K, and angiotensin.

The chelating moieties may be represented by the general formula I:

where R¹, R², and R³ independently are selected from the groupconsisting of H, lower alkyl, substituted lower alkyl, cycloalkyl,substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkaryl, and a protecting group thatcan be removed under the conditions of peptide synthesis. At least oneof R¹, R², or R³ must be H. R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ independentlyare selected from the group consisting of H, lower alkyl, substitutedlower alkyl, aryl, and substituted aryl. R⁴ and R⁶ together also mayoptionally form a direct bond, and R⁸ and R⁹ together or R⁷ and R⁹together also may form a cycloalkyl or substituted cycloalkyl ring. NR¹⁰is located at the N-terminus of the peptide to which the chelator isattached, or is located on an amino acid side chain of that peptide.When R¹, R², R⁵, or R⁶ bears a heteroatom substituted function, theheteroatom also may be used to carry out additional peptide couplingreactions.

Examples of lower alkyl include, but are not limited to, straight orbranched chain C₁–C₆ alkyl groups, such as a methyl, ethyl, n-propyl,i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, andn-hexyl. Cycloalkyl includes C₃–C₆ cycloalkyl, such as cyclohexyl.Heterocycloalkyl includes tetrahydrofuran, tetrahydropyran, pyrrolidine,and piperidine. Heteroaryl includes pyrrolyl, furanyl, thienyl,imidazolyl, oxazolyl, oxazolylthio, thiazolyl, pyrazolyl pyrrolidinyl,pyridinyl, pyrimidinyl, morpholinyl, and piperizinyl. Aryl includesC₆–C₁₂ aryl such as phenyl, α-naphthyl, or β-naphthyl.

Alkaryl includes: C₆–C₁₂arylC₁–C₆alkyl, such as phenylC₁–C₆alkyl, or α-or β-naphthylC₁–C₆alkyl, such as benzyl, phenylethyl, phenylpropyl,phenylbutyl, phenylpentyl, α- or β-naphthylmethyl, napthylethyl,naphthylpropyl, naphthylbutyl, or naphthylpentyl.

Examples of substituent groups include: C₁–C₆ alkoxy, for example,methoxy, ethoxy, propoxy; C₁–C₆ alkylthio, for example methylthio,ethylthio, propylthio; C₆–C₁₂arylC₁–C₆alkoxy, for example phenylC₁–C₆alkoxy such as benzyloxy; aralkylthio, for example phenylC₁–C₆alkylthiosuch as benzylthio; amino, substituted amino, for exampleC₁–C₆alkylamino such as methylamino, ethylamino; C₆–C₁₂arylC₁–C₆alkyl,such as phenylC₁–C₆ alkyl for example benzyl; C₆–C₁₂aryl such as phenyl;C₃–C₈ cycloalkyl such as cyclohexyl; and C₃–C₈ cycloalkylC₁–C₆alkyl suchas cyclohexylmethyl.

Preferred embodiments of the invention include compounds where R² is H,methyl, or phenyl, and where R⁸ and R⁹ are methyl. Other preferredembodiments are where R¹, R³, R⁵, R⁷ and R¹⁰ are H.

The peptides may be synthesized using differentially protected bis-aminoacid derivatives in which either amino function can be selectivelydeprotected. These derivatives are introduced into a growing peptidechain during peptide synthesis by conventional peptide couplingmethodology. One of the amino functions is then selectively deprotected,allowing subsequent coupling of either all or a part of a chelatingmolecule, or addition of further amino acid residues to continue thepeptide synthesis. Peptide synthesis can be continued by coupling at theα-amino group, leading to a peptide with a conventional amide backbone,or at the side-chain amino group to produce a peptide whose amidebackbone is interrupted by the side chain structure. Alternatively, thefree amino function can be used to cyclize onto a reactive functionalitylocated elsewhere in the peptide, thereby producing a cyclic peptide.Suitable bis-amino acids will be readily apparent to the skilledpractitioner, and include lysine, ornithine, and 2,3-diaminopropionicacid (amino-serine). Alternatively, the chelating moiety may beintroduced at the end of peptide synthesis by coupling the chelatingmoiety to the deprotected N-terminus of the resin-bound peptide. Thechelating moiety may be added as a complete unit, in protected orunprotected form, or may be synthesized in stepwise fashion to constructthe complete chelating structure.

Bis-amino acids used in the present invention may be generallyrepresented by the formula: ZHN—CH(—R—NHY)—CO₂H where R is (CH₂)_(n) or(CH₂)_(n)—X—(CH₂)_(n) where X is a heteroatom such as O, S, or N andn=1–20. Alternatively the hydrogen atoms of the CH₂ groups can bereplaced with lower alkyl, substituted lower alkyl, or alkenyl groups,or cyclic or heterocyclic rings such as cyclohexane, benzene, andpiperidine, or other groups well known to the skilled artisan. Thesubstituents Z and Y independently can be H, N, lower alkyl, substitutedlower alkyl, aryl, or substituted aryl.

If peptide synthesis is continued, selective deprotection of the secondamino group of the bis-amino acid can be accomplished at any pointduring the peptide synthesis to introduce the chelating moiety. Thecomplete chelating moiety can be synthesized prior to coupling to thepeptide, or it can be synthesized by sequentially coupling segments tothe peptide. Once assembly of the entire peptide/chelator structure iscomplete, cleavage, deprotection, and purification affords the desiredpeptide derivative. This derivative is then labeled with a radiometalfor use in radiodiagnostic and radiotherapeutic applications.

Alternatively, if all or part of the chelating molecule is coupled tothe deprotected amino group first, the second step is to deprotect theother amino group and continue with the peptide synthesis. If only partof the chelator moiety is coupled to the peptide at this stage, thesynthesis of the chelator can be finished at any point during or aftersynthesis of the peptide chain by appropriate deprotection and couplingreactions. Final cleavage, deprotection and purification steps onceagain yield the pure peptide derivative, which is then radiolabeled asbefore.

Attachment of the chelator to the peptide prior to cleavage from theresin results in reduced formation of cross-linked products even whenmultidentate activated chelators such as DTPA-dianhydride are used.

Preparation of cyclic peptides is achieved by selective deprotection oftwo compatible functional moieties at specified positions of the peptidesequence, followed by cyclization between the compatible moieties.Cyclization can be achieved between any two points of the peptidesequence, including between the N- and C-termini, between a terminus andan internal functional group within the peptide sequence, or between twointernal functional groups. Cyclization can be achieved using eithersolution-phase or solid-phase peptide syntheses, but is preferablycarried out using solid-phase techniques.

The deprotection and cyclization can be carried out at any point duringthe synthesis of the peptide prior to the final deprotection reactions.For example, the entire protected peptide sequence can be prepared priorto the cyclization, or the cyclization can be carried out on a protectedpeptide intermediate, followed by completion of the synthesis.Similarly, the cyclization can be carried out either before or after allor part of the metal chelating moiety is coupled to the peptide. Use ofa photocleavable or other resin known to those skilled in the art on asolid phase peptide synthesizer also allows release of a protectedpeptide from a solid support, with subsequent solution phase selectivedeprotection and cyclization. Alternatively, the side chains to becyclized can be selectively deprotected prior to cleavage from theresin, and the cyclization carried out in solution phase.

Reactions involving the C-terminus of the peptide, including but notlimited to cyclization reactions, may be accomplished through therelease of a protected peptide from the resin in the manner describedabove. Alternatively, the growing peptide chain may be attached to theresin via the side chain of a residue and the C-terminal carboxyl groupsuitably protected. When a reaction with the C-terminal carboxyl groupis desired, it is selectively deprotected and the reaction allowed toproceed. In the case of cyclization reactions, the deprotection of theC-terminus can be accomplished before, during, or after the selectivedeprotection of the compatible reactive group.

The radiometal chelating peptides of the present invention stably retainradionuclide in blood and other bodily fluids and tissues. Both thereagents and the conditions in the present method are greatly simplifiedover those in the prior art, and the labeled peptides are particularlysuitable for radiodiagnostic and radiotherapy applications usingtechnetium or rhenium labeling.

The approach outlined above allows the placement of a radiometal-bindingmoiety anywhere in a peptide sequence. Placing the chelating moiety onan amino acid side-chain, either directly or via a spacer group, ratherthan on the N-terminus of a peptide, has the added advantage ofspatially distancing the metal complex from the peptide backbone,thereby minimizing the effect of the metal complex on the peptideconformation. This also allows the N-terminus of the peptide to be usedfor cyclizing the peptide, if necessary.

It is known that peptide conformation is greatly influenced by chargeand hydrophilic/hydrophobic interactions, and it is therefore importantto consider these variables when designing a chelating ligand to be usedin peptides. It is preferred that a variety of chelating complexes ofvarying charge and hydrophilicity and containing spacer groups ofvarious lengths are prepared and tested to select the metal-complexedpeptide that displays the optimum combination of target selectivity,pharmacokinetics, and chelate stability. The skilled artisan willappreciate that such testing is routine in the art.

The radiolabeled peptides of the present invention bind specifically toa diseased cell or tissue that exhibits both a high receptor density andhigh affinity for the peptide. The radioactivity of the radionuclideallows diagnosis and/or treatment of the tumor or diseased tissue. Theinvention also includes pharmaceutical compositions comprising aneffective amount of at least one of the radiolabeled peptides of theinvention, in combination with a pharmaceutically acceptable sterilevehicle, as described, for example, in Remington's PharmaceuticalSciences; Drug Receptors and Receptor Theory, 18th ed., Mack PublishingCo., Easton, Pa. (1990). The invention also includes kits for labelingpeptides which are convenient and easy to use in a clinical environment.

A. Design and Synthesis of Linear Peptides Incorporating ChelatingMoieties

(i) In General

The peptides of the invention contain radiometal-chelating amino acidderivatives that are characterized by the presence of at least one thiolor thiocarbonyl group, and at least one nitrogen present as either atertiary amine, a hydrazone, or a secondary amide or hydrazide. Thesulfur and nitrogen atoms are suitably disposed to form a multidentateligand capable of tightly and preferentially binding a metal ion. Themultidentate ligand may also contain a spacer group that serves toseparate the chelated metal from the rest of the peptide. The metal ionis preferably a reduced radionuclide, and in a preferred embodiment is^(99m)Tc, ¹⁸⁶Re, or ¹⁸⁸Re.

The invention also provides a method for placing ligands for othermetals at any point in a peptide sequence. These ligands can beintroduced intact, for example DTPA, or as fragments. In this wayligands for other metals of medical interest including, but not limitedto, In, Ga, Y, Cu, Pt, Mn, Gd, Au, Ag, Hg, and Lu can be placed in apeptide targeting sequence.

The method allows the introduction of any metal chelate or chelatefragment that is suitably protected for peptide synthesis. The methodalso provides a method for the introduction of base-sensitive ligandderivatives that can be placed at any point in the peptide sequence aslong as it is introduced at the end of the synthesis. An example is thesynthesis of a ligand attached to the side chain of lysine using thefollowing ligand fragment Trityl-S—COCH₂N(Boc)CH₂CO₂H. The S-Tritylester will be sensitive towards base so it would not be possible toplace this ligand fragment on the peptide at an earlier point in thesynthesis.

Each of the chelating moieties of the invention can be prepared bymethods well known to the skilled practitioner in the art of organicsynthesis. The chelating moieties are constructed from subunits that arelinked together by simple coupling or condensation reactions, such asthe condensation of an amino, hydrazino, or hydrazido function with anactivated carboxyl group, coupling of hydrazines with aldehydes, orreductive amination reactions between amines and aldehydes. As usedherein the term “condensation” is intended to encompass reactions thatcouple together subunits of the chelating moiety, and thus encompassesreactions such as reductive amination in addition to reactions thatconform to the classical definition of a condensation reaction.

Following a condensation reaction, additional functional groups on thesubunit may be deprotected to allow additional condensation reactions.For example, a second subunit carrying a free carboxyl group and aprotected amino function can be condensed with an amino, hydrazino, orhydrazido function on a first subunit to produce a larger, suitablyprotected fragment of the metal binding ligand. The amino function onthe second subunit moiety can then be deprotected and further coupled toa third subunit. As used herein, the term “fragment” is intended toencompass a subunit or assembly of subunits comprising all or part ofthe metal binding ligand.

Methods of activating carboxyl groups for such condensation reactionsare well known to those of skill in the art of organic synthesis andpeptide synthesis, and include the use of active esters and ofcarbodiimide and phosphoryl azide coupling agents. Suitable protectinggroups are used for protecting functions on the subunits when thereactivity of the functions is incompatible with a reaction used to jointhe subunits or with reactions used for synthesis of the peptide chain.Protecting groups for mercapto, amino and carboxylic acid functions arewell known in the art. See, for example, Greene, PROTECTIVE GROUPS INORGANIC SYNTHESIS (Wiley Interscience, N.Y., 1981). The subunits used toconstruct the chelate are either readily prepared by methods well knownin the art, or are commercially available from suppliers such asAdvanced ChemTech (Lexington, Ky.), Milligen (Burlington, Mass.),Applied Biosystems (Foster City, Calif.), or Aldrich Chemical Corp.(Milwaukee, Wis.).

The condensation reactions used to link together the chelator subunitscan either be carried out prior to peptide synthesis, or duringsynthesis of the peptide sequence. When the amino acid derivative isassembled from its subunits prior to peptide synthesis, α-amino andα-carboxyl functions must be suitably protected in a manner that issubsequently compatible with selective deprotection and activation ofthese functionalities for peptide synthesis. Examples of such protectinggroups are well known in the art, and include thefluorenemethyloxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz),^(t)butoxycarbonyl (Boc), allyloxycarbonyl (aloc), 4-methoxytrityl(mtt), and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde)groups for amino protection. Groups for carboxyl protection include themethyl (Me), benzyl (Bn), ^(t)butyl (^(t)Bu), and allyl esters,respectively.

The amino and carboxyl protecting groups must be selected such that eachgroup can be selectively deprotected in the presence of the other. Suchprotecting moieties are said to be orthogonal. The requirement thatorthogonal protecting groups be used precludes, for example, use of theCBZ group for protection of the amino function in the presence of acarboxyl group protected as a benzyl ester. See Greene, supra. In apreferred embodiment the α-amino group is protected as an Fmoc group,and the α-carboxyl group is a methyl ester. The thiol protecting groupused in the compounds of the invention can be any organic or inorganicgroup which is readily removed under mild conditions to regenerate thefree sulfhydryl in the presence of the peptide without substantiallyaltering the activity of the protein. Suitable protecting groups arelisted in Greene, supra, pp. 193–217. Examples of suitable protectinggroups include substituted and unsubstituted trityl groups, thiolesters, thiocarbamates and disulfides. In a preferred embodiment thethiol protecting group is a trityl group or a 4-methoxytrityl group.Those skilled in the art are familiar with the procedures of protectingand deprotecting thiol groups. For example, benzoate thioesters may bedeprotected under mild and selective conditions using hydroxylamine.Once assembly of the protected chelating moiety is complete, theα-carboxy function is deprotected and coupled to the amino terminus ofthe peptide chain using conventional methods of peptide synthesis. See,Bodanszky et al., THE PRACTICE OF PEPTIDE SYNTHESIS (Springer Verlag,Heidelberg, 1984).

When the metal-chelating amino acid derivative is assembled from itssubunits during peptide synthesis, the peptide chain is assembled byconventional solution phase or, preferably, solid phase synthesis untilthe point where the derivative is to be incorporated. The differentiallyprotected bis-amino acid is then coupled to the amino terminus of thepeptide chain. Subsequent selective deprotection of one of the aminogroups of the derivative allows either peptide synthesis or chelatorsynthesis to continue.

If the α-amino function is deprotected first, all or part of theremaining amino acid residues are then coupled to the peptide chain inthe conventional manner. The side chain amino function of the derivativeis then deprotected, and the chelating moiety is assembled as describedabove. The complete peptide can then be deprotected and purified bystandard methods.

If the side chain amino function is deprotected first, all or part ofthe chelating moiety is then assembled as described above, followed bydeprotection of the α-amino group. Peptide synthesis is completed in theconventional manner as described above.

Once peptide synthesis is complete the fully protected peptide isdeprotected and purified. Methods for deprotection and purification ofsynthetic peptides are well known in the art. See, for example,Bodanszky, supra. If the peptide was synthesized by solid phasetechniques the peptide must also be cleaved from the resin used as thesolid support for the synthesis. Methods for achieving this cleavagealso are well known in the art. Methods for purifying synthetic peptidessuch as those of the present invention also are well known to those ofskill in the art. Such methods include, for example, ion exchange, gelfiltration chromatography, and reversed phase high pressure liquidchromatography (RP-HPLC). In a preferred embodiment of the invention thepeptide is purified by RP-HPLC using a preparative-scale octadecylsilane(C18) silica column packing, eluting with a gradient of acetonitrile in0.1% trifluoroacetic acid (TFA). The purity of the peptide can beconfirmed by standard methods such as analytical RP-HPLC or capillaryelectrophoresis. The identity of the peptide can be confirmed by NMRspectroscopy or, in a preferred embodiment of the invention, by massspectrometry.

As noted above, it is important that the chelating moiety does notinterfere with peptide binding to the appropriate receptor. Determiningthe residues within the peptide that can be replaced withoutdeleteriously affecting receptor binding can be carried out in asystematic and straightforward way by preparing a series of peptides inwhich each successive residue is replaced with, for example, alanine,(an “alanine scan”). The alanine-substituted peptides then are screenedfor biological activity. Modern peptide synthesizers make synthesis ofpeptides in this way quite straightforward, and screening of a largenumber of peptides is routine for the skilled artisan. Retention of highreceptor binding affinity in a peptide containing such an alaninesubstitution denotes that the substituted amino acid is less importantfor receptor binding, and indicates a position where the metal-bindingresidue may be placed. Synthesis of peptides wherein a chelator isplaced in each of these positions is then straightforward, and routinescreening establishes the optimal position for the chelator.

As set forth above, a wide variety of peptides containing metal bindingligands may be prepared using the methods of the present invention.Additional methods of preparing metal chelating peptides using themethods of the claimed invention will be apparent to the skilledartisan. Specific applications using these methods are set forth belowto further-exemplify the invention, but it will be appreciated thatthese examples are merely illustrative and are not meant to limit thescope of application of the invention.

(ii) Preparation of Chelating Moieties

In a preferred embodiment, the chelator contains a thiol group togetherwith a thiosemicarbazide or thiosemicarbazone group, and can berepresented by the general formula I:

where R¹, R², and R³ independently are selected from the groupconsisting of H, lower alkyl, substituted lower alkyl, cycloalkyl,substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkaryl, and a protecting group thatcan be removed under the conditions of peptide synthesis. At least oneof R¹, R², or R³ must be H. R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ independentlyare selected from the group consisting of H, lower alkyl, substitutedlower alkyl, aryl, and substituted aryl. R⁴ and R⁶ together also mayoptionally form a direct bond, and R⁸ and R⁹ together or R⁷ and R⁹together also may form a cycloalkyl or substituted cycloalkyl ring. NR¹⁰is located at the N-terminus of the peptide to which the chelator isattached, or is located on an amino acid side chain of that peptide.When R¹, R², R⁵, or R⁶ bears a heteroatom substituted function, theheteroatom also may be used to carry out additional peptide couplingreactions.

Although an understanding of the mechanism of metal binding by thechelating moieties is not necessary for practicing the invention, andwithout wishing to be bound by any theory, it is believed that the metalis bound to the chelator via the two sulfur atoms plus two nitrogenatoms. It is hypothesized that the metal-binding nitrogens are theα-nitrogen of the β-thiol-containing amino acid and thethiocarbazide/thiocarbazone nitrogen distal to the thiocarbonyl group.When the metal is reduced radioperrhenate or reduced radiopertechnetate,the two sulfur and two nitrogen atoms provide four coordinationpositions on the metal.

These compounds may be prepared by methods that are well known in theart of organic synthesis. Thus, for example, compounds having formula Imay be prepared by amide coupling between an α-carboxyl-protected aminoacid moiety having a protected or unprotected β-thiol-containing sidechain (a “cysteine-type amino acid”), and a carboxyl-containingthiosemicarbazone or thiosemicarbazide moiety. This coupling can becarried out using well-known methods, such as carbodiimide-mediatedcoupling. Deprotection of the α-carboxyl group of the amino acid allowsamide coupling of the chelating moiety to an amino side chain, or theamino terminus, of the peptide. Alternatively, an N- and S-protectedcysteine-type amino acid having a β-thiol containing side chain mayfirst be coupled to the peptide, followed by N-deprotection and amidecoupling to a carboxyl-containing thiosemicarbazone or thiosemicarbazidemoiety.

Cysteine-type amino acids may be prepared by standard methods of aminoacid synthesis. The configuration at the α-carbon may be (R) or (S), orthe amino acid may be racemic. Similarly, the configuration at theβ-carbon, when asymmetrically substituted may be (R), (S), or (R/S). Theamino acid is protected for subsequent coupling reactions using standardmethods.

Thiosemicarbazones may be prepared by the condensation of semicarbazideswith carbonyl compounds. Reduction of the thiosemicarbazones with, forexample, sodium borohydride, provides substituted thiosemicarbazides. Ina preferred embodiment, a thiosemicarbazide is reacted with glyoxylicacid to form the corresponding thiosemicarbazone, which optionally maybe reduced to form a substituted thiosemicarbazide.

Many thiosemicarbazides are commercially available from, for example,Aldrich Chemical Company, Milwaukee, Wis. Other thiosemicarbazides maybe prepared by the reaction of a hydrazine with, for example, anisothiocyanate. Asymmetrically substituted hydrazines also arecommercially available, or may be prepared by nitrosation of amines tothe nitrosamine, followed by reduction to the hydrazine. Isothiocyanatesmay be prepared by reaction of an amine with thiophosgene. Other methodsof preparing thiosemicarbazides are well known to the skilled artisan.

In some instances, it is found that thiosemicarbazides have lowsolubility in the solvents used for coupling to the cysteine-like aminoacid. In such instances, the coupling can be carried out using thethiosemicarbazone, followed by coupling to the peptide. Thethiosemicarbazone then may be reduced to the thiosemicarbazide usingsodium borohydride or another suitable reducing agent. When thethiosemicarbazide is sufficiently soluble to be used directly, theα-nitrogen must first be protected prior to coupling. Suitableprotecting groups are well known in the art and include Boc and Cbzgroups.

(iii) Linear VIP Receptor Targeting Agents

Naturally occurring VIP has the sequence:

-   HSDAVFTDNYTRLRKQMAVKKYLNSILN-NH₂(SEQ ID NO:2)    An alanine scan has revealed several residues whose replacement with    alanine does not greatly affect receptor binding. These residues    include Lys-15, Gln-16, Val-19, Lys-21, Asn-24, Ser-25 and the N-    and C-termini. These locations are possible sites for the attachment    of a metal binding ligand according to the present invention.

Chelating derivatives based on attachment of the metal binding ligand atthese positions include, but are not limited to, those with a metalbinding moiety attached, either directly or via a spacer group, to thepharmacophore via the side chain amine of a lysine or other bis-aminoacid residue. Specific chelating derivatives of this general structureinclude, but are not limited to:

-   MaGCγAbuHSDAVFTDNYTRLRKQMAVKKYLNSILN-NH₂ (SEQ ID NO:2)-   AcCGCHSDAVFTDNYTRLRKQMAVKKYLNSILN-NH₂ (SEQ ID NO:22)-   KPRRPYTDNYTRLRK(PtscGC)QMAVKKYLNSILN-NH₂ (SEQ ID NO:3)-   MaGCγAbuVFTDNYTRLRKQMAVKKYLNSILN-NH₂ (SEQ ID NO:4)-   AcCGCVFTDNYTRLRKQMAVKKYLNSILN-NH₂ (SEQ ID NO:23)-   MaGCγAbuYTRLRKQMAVKKYLNSILN-NH₂ (SEQ ID NO:5)-   HSDAVFTDNYTRLRK(PtscGC)QMAVKKYLNSILN-NH₂ (SEQ ID NO:2)-   HSDAVFTDNYTRLRK(Dtpa)QMAVKKYLNSILN-NH₂ (SEQ ID NO:2)-   HSDAVFTDNYTRLRK(AGC)QMAVKKYLNSILN-NH₂ (SEQ ID NO:2)-   where Ma is mercaptoacetic acid,-   PtscG is 2-(4-phenyl-3-thiosemicarbazidyl)acetic acid or    PhNHCSNHNHCH₂ CO₂H,-   γAbu is γ-aminobutyric acid, and-   in K(PtscGC), the parentheses denote that enclosed amino acids are    attached to the epsilon. amine of lysine and the first amino acid    attached is C followed by PtscG.

In each of the compounds described above, the chelating moiety may bereplaced by a chelator of the general formula I, as described above.

(iv) Linear LHRH Receptor Targeting Agents

Naturally occurring LHRH has the sequence:

-   <GHWSYGLRPG-NH₂ (SEQ ID NO:24)    where <G is pyroglutamic acid. It is further known that the bicyclic    peptide AcNal_(d) Cpa_(d) W_(d) (cyclo4–10)D(cyclo5–8)ER_(d)    LKPDap-NH₂ (SEQ ID NO:25) (where W_(d) indicates that the D isomer    of the amino acid was used, Nal is 2-naphthylalanine, Cpa is    4-chlorophenylalanine and Dap is 2,3-diaminopropionic acid) binds to    the LHRH receptor. See Bienstock et al. J. Med. Chem. 36:3265    (1993). It is also known that the side chain of position 6 of LHRH    is very bulk tolerant. See Barbacci et al. J. Biol. Chem. 270:9585    (1995). This location is a possible site for the attachment of a    metal binding ligand according to the present invention.

Linear chelating derivatives based on attachment of the metal bindingligand at this position include, but are not limited to, those with ametal binding moiety attached, either directly or via a spacer group, tothe pharmacophore via the side chain amine of a lysine or otherbis-amino acid residue. Specific linear chelating derivatives of thesegeneral structures include, but are not limited to:

-   <GHWSYK(MaGC)LRPG-NH₂ (SEQ ID NO:6)-   <GHYSLK(MaGC)WKPG-NH₂ (SEQ ID NO:7)-   <GHWSYK(Ma-azaGC)LRPG-NH₂ (SEQ ID NO:6)-   <GHYSLK(PtscGC)WKPG-NH₂ (SEQ ID NO:7)-   <GHYSLK(PtscGDap)WKPG-NH₂ (SEQ ID NO:7)-   <GHWSYK_(d)(MaGC)LRPG-NH₂ (SEQ ID NO:13)-   <GHYSLK(azaGGC)WKPG-NH₂ (SEQ ID NO:7)-   <GHWSYK(iECG)LRPG-NH₂ (SEQ ID NO:6)-   <GHWSYK_(d)(MtaGC_(a))LRPG-NH₂ (SEQ ID NO:13)-   <GHYSLK(iECiD)WKPG-NH₂ (SEQ ID NO:7)-   <GHYSLK(DiGlyGDap)WKPG-NH₂ (SEQ ID NO:7)-   <GHYSLK(iDGDap)WKPG-NH₂ (SEQ ID NO:7)-   <GHWSYK(MtaGC)LRPG-NH₂ (SEQ ID NO:6)-   <GHWSK(MaGC)W_(d)LRPG-NH₂ (SEQ ID NO:26)-   <GHWSYK_(d) (MtaGDap)LRPG-NH₂ (SEQ ID NO:13)-   <GHWSYK_(d) (PtscGC)LRPG-NH₂ (SEQ ID NO:13)-   <GHWSYK_(d) (E)LRPG-NH₂ (SEQ ID NO:13)-   <GHWSYK_(d) (MtscGC)LRPG-NH₂ (SEQ ID NO:13)-   <GHWSYK_(d) (Mta(hqss)GDap)LPG-NH₂ (SEQ ID NO:8)-   AcNal_(d) Cpa_(d) W_(d) SRK_(d) (MaGC)LRPA_(d)-NH₂ (SEQ ID NO:8)-   Nal_(d) Cpa_(d) W_(d) SRK_(d) (PtscGC)LRPA_(d)-NH₂ (SEQ ID NO:8)-   AcNal_(d) Cpa_(d) W_(d) SRK_(d) (MaFC)LRPA_(d)-NH₂ (SEQ ID NO:8)-   AcNal_(d) Cpa_(d) W_(d) SRK_(d) (azaGFC)LRPA_(d)-NH₂ (SEQ ID NO:6)-   where:-   <G is pyroglutamic acid,-   Ma is mercaptoacetic acid-   azaG is azaglycine or H₂NNHCH₂ CO₂H,-   PtscG is 2-(4-phenyl-3-thiosemicarbazidyl)acetic acid or    PhNHCSNHNHCH₂ CO₂H,-   Dap is 2,3-diaminopropionic acid-   iD is an aspartic acid coupled via the side chain carboxyl group,-   iE is a glutamic acid coupled via the side chain acid group,-   DiGly is HOOCCH₂ NHCH_(d) COO—,-   Mta(hqss) is S-(2,5-dihydroxyphenyl-S-methyl) sulfoniumacetyl-   C_(a) is an Acm protected cysteine-   Mta is the methylthioether of mercaptoacetic acid,-   Nal is 2-naphthylalanine,-   Cpa is 4-chlorophenylalanine,-   in K_(d), the subscript d denotes that the D isomer was used, and-   in K(MaGC), the parentheses denote that enclosed amino acids are    attached to the ε amine of lysine and the first amino acid attached    is C followed by G and ending in Ma.

Additionally, complexes of these peptides with non-radioactive metalsmay be prepared. Such complexes include:

-   <GHWSYK(MaGC)LRPG-NH₂ ReO-   <GHYSLK(MaGC)WKPG-NH₂ ReO (SEQ ID NO:7)-   <GHYSLK_(d)(MaGC)LRPG-NH₂ReO (SEQ ID NO:27)

In each of the compounds described above, the chelating moiety may bereplaced by a chelator of the general formula I, as described above.

(v) Linear α-MSH Receptor Targeting Agents

Naturally occurring α-MSH has the sequence:

-   Ac-SYSMEHFRWGKPV-NH₂ (SEQ ID NO:28).

It had previously been shown that the cyclic peptide NleDHF _(d) RWK-NH₂(SEQ ID NO:1) (where Nle is norleucine and F_(d) indicates D-Phe) has ahigh affinity for the α-MSH receptor and is known to be relativelystable in-vivo. See Al-Obeidi et al. J. Amer. Chem. Soc. 111:3413(1989); Haskell-Luevano et al. J. Med. Chem. 39:432 (1996). Theunderlined portion indicates those residues within the cyclized portionof the peptide, and also the termini of the cyclic structure, i.e. thepeptide is cyclized by an amide bond from the side chains of asparticacid and lysine.

Linear chelating derivatives based upon the structures of these knownα-MSH receptor binding peptides include those with a chelatingderivative attached to the N-terminus of the peptide, either directly orvia a spacer group, such as γ-amino butyric acid (γ-Abu). Specificlinear chelating derivatives with this general structure include, butare not limited to:

-   MaGCγAbuSYSNleDHF_(d) RWK-NH₂, (SEQ ID NO:9) and-   MaGCγAbuSYSNleDHF_(d) R_(n) WK-NH₂ (SEQ ID NO:29)    where γ-Abu is γ-aminobutyric acid and R_(n) is a nitrated arginine    residue.

In each of the compounds described above, the chelating moiety may bereplaced by a chelator of the general formula I, as described above.

B. Design and Synthesis of Cyclic Peptides Incorporating Chelating AminoAcid Derivatives

(i) In General

The process of preparing a cyclic metal-chelator/peptide complex isanalogous to that described above for linear peptides, except that atsome point during or subsequent to synthesis of the peptide chaincyclization is carried out. The cyclization can be between any twofunctional groups on the peptide such as the peptide termini or aminoacid side chains. The cyclization can be achieved by disulfide orsulfide formation, or preferably by lactam formation. Site-selectivecyclization requires selective deprotection of two functional groups onthe peptide. For lactam formation this requires using an amino and acarboxyl protecting group that can be deprotected in the presence ofother amino and carboxyl protecting groups. This task is made moredifficult when the peptide synthesis also requires that selectivedeprotection be achieved between these other protecting groups.Accordingly, such a sophisticated protecting group strategy hasheretofore proved difficult to achieve in practice.

The methods of the present invention allow both cyclization and couplingof the chelator moiety to the peptide to be achieved at any point duringpeptide synthesis. Use of appropriate protecting groups allows synthesisof the peptide, assembly of the ligand and cyclization of the peptide tobe achieved in any order that is desired. This approach is moreefficient than either solid-phase methods which cyclize the peptide offthe resin or methods that attach ligands in solution following synthesisof the cyclic peptide.

The methods of the present invention may be used in solution phase, butpreferably are carried-out using an automated solid-phase peptidesynthesizer. Using a multi-well automated synthesizer allows a largenumber of peptides, differing in the point of attachment of the chelatormoiety or in the site of cyclization, to be prepared simultaneously.These so-called “combinatorial libraries,” wherein the ligand containingpeptides are deprotected while still attached to the solid support, canbe reacted with the appropriate-metal to form complexes and thenscreened in an appropriate bioactivity assay to select the compoundhaving the optimally desired characteristics of receptor binding andstability.

Combinatorial synthesis can be carried out in “split” syntheses or by“parallel” syntheses. In split synthesis, synthetic peptideintermediates bound to beads are subdivided into different groups foraddition of the next amino acid in each successive step. After each stepthe beads are divided into different groups for the next reaction. Inparallel synthesis, different compounds are synthesized in differentreactions vessels, such as the wells of a peptide synthesizer. Splitsynthesis provides small quantities of large numbers of compounds,whereas parallel synthesis provides larger quantities of a smallernumber of compounds.

Combinatorial synthesis also requires that each individual compound belabeled in some way in order that it might be identified in thescreening step. Various means of labeling compounds for this purpose areknown in the art. For example, inert halogenated aromatic compounds areused as labels that can be identified by gas chromatography. See Borman,Chem. Eng. News 74:29 (1996) which is hereby incorporated herein in itsentirety.

In a preferred embodiment of the invention cyclization is achieved bylactam formation. Most preferably the amino function is protected usingan aloc group, and the carboxyl function is protected as an allyl ester.This allows simultaneous deprotection of the amino and carboxy functionsusing Pd(PPh₃)₄ in the presence of a nucleophile for the allyl group.The nucleophile typically used is tri-n-butyl tin hydride (^(n)Bu₃SnH).

Prior to the present invention it was known that amines would react withallyl ions under Pd⁰ catalysis. See, for example, Roos et al., J. Org.Chem. 60:1733 (1995) and Heck, PALLADIUM REAGENTS IN ORGANIC SYNTHESES,Academic Press, 1995, pp. 122–131. The present inventors found that thiscaused a problem for simultaneous deprotection of allyl-protected aminoand carboxyl functions because of the side reaction wherein the newlydeprotected amino function was alkylated with the allyl group. It wasfound, however, that addition of piperidine as an allyl scavenger duringthe Pd-catalyzed aloc cleavage reaction inhibited this unwantedside-reaction. This allowed deprotection of, for example, aspartic acidand lysine side chains selectively and simultaneously while greatlyreducing formation of the undesired N^(ε)-allyl lysine. Those skilled inthe art will recognize that other primary or secondary amines will alsobe suitable allyl scavengers.

This method is useful and advantageous because it is compatible withFmoc based peptide synthesis. It allows preparation of cyclic peptideson HMP and PAM resins where both the Boc or Fmoc side chain protectioncan be used in addition to Aloc side chain protection. This techniquefurthermore allows synthesis of cyclic peptides containing ametal-binding ligand attached to the side chain of an amino acid at anypoint in a peptide chain. Thus three orthogonal nitrogen protectinggroups are used: one for building the peptide chain, such as Fmoc orBoc; a second for attaching a metal-binding ligand to a side chain of abisaminoacid such as 4-methyltrityl(mtt) or1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde); and a third,such as Aloc, for achieving side-chain to side-chain cyclization. Otherprotecting groups, such as Boc, Cbz, ^(t)Bu and benzyl groups can beused for protection of other side-chain amino and carboy functions thatare not deprotected until the peptide synthesis is finished.Combinations of these protecting groups allow the use of Rink, Wang,Merrifield, PAM and HMP-type resins for solid-phase peptide synthesis.

Cleavage of the peptides from the resin can be accomplished withtrifluoroacetic acid (for Fmoc-based syntheses) or HF or trimethylsilyltrifluoromethanesulfonate (for Boc-based syntheses).

The cyclic peptides of the invention are from 4 to 100 residues long,and have up to 5 metal chelating groups. The peptides can containcyclized regions that are between 2 and 60 amino acids in length, andcan contain more than one cyclic portion.

Cyclization is preferably between amino and carboxy amino acid sidechains to form lactam bridges, but may also be between a side chainamine and the carboxy terminus to form a lactam bridge, between a sidechain acid and the N-terminal amine to form lactam, between thiols toform disulfide bridges, between hydrazines and esters to formhydrazides, between hydrazines and aldehydes to from hydrazones, betweena thiol and a suitable leaving group to form a sulfide, or between ahydroxyl group and another suitable leaving group to form an ether. Whenmore than one cyclic region is present in the compound, the bridges inthe cyclic regions may be of the same or different types.

When a cyclic disulfide is to be formed, the peptide of interest ispreferably synthesized using Acm protection on thiols and alocprotection on amino side chains. The aloc is cleaved and the chelatingligand is coupled to the peptide. The Acm group is then cleaved and thedisulfide cyclized using thallium trifluoroacetate. Alternatively, thethiols can be protected using S-trityl groups, and the cyclization canbe carried out in solution after cleavage from the resin.

Preparation of a cyclic sulfide may be achieved by, for example,cyclization of a thiol onto an α-haloamido function present on an amineside chain. Thus, for example, the peptide may be synthesized with Fmocprotection on the N-terminus and S-trityl protection on the thiol. Analoc-protected lysine side chain is then deprotected and the chelator iscoupled to the lysine as described above. The Fmoc is cleaved andreacted with chloroacetyl chloride or an equivalent reagent. If an acidstable resin such as the photocleavable BromoWang resin, Wang, J. Org.Chem. 41:3258 (1976), is used the thiol protecting group is removed andthe peptide is cyclized on the resin.

Cyclic sulfides between side chain residues are prepared by using asuitable protecting group on the N-terminus of the peptide that allowsselective deprotection at two differentially protected amino sidechains. For example, the N-terminus can be protected as an acetyl or Bocgroup, and the side chains can be protected as Fmoc and aloc groups.Following peptide synthesis, one amino acid side chain (for example,Fmoc) is selectively deprotected and coupled with the chelate moiety asdescribed above. Another amino side chain (for example aloc or Dde) isthen deprotected and coupled to a sulfide electrophile such ashaloacetyl or maleimide. The protecting group on the sulfur of interestis cleaved and cyclization is carried out on the resin using a basecatalyst. Alternatively, the peptide can be cleaved and the cyclizationcan be carried out in solution. As set forth above, a wide variety ofcyclic peptides may be prepared using the methods of the presentinvention. Additional methods of preparing cyclic metal chelatingpeptides using the methods of the claimed invention will be apparent tothe skilled artisan. Specific applications using these methods are setforth below to further exemplify the invention, but it will beappreciated that these examples are merely illustrative and are notmeant to limit the scope of application of the invention.

(ii) Cyclic α-MSH Receptor Targeting Agents

Naturally occurring α-MSH has the sequence Ac-SYSMEHFRWGKPV-NH₂ (SEQ IDNO:28). It had previously been shown that the cyclic peptide NleDHF _(d)RWK-NH₂ (SEQ ID NO:1) (where Nle is norleucine and F_(d) indicatesD-Phe) has a high affinity for the α-MSH receptor and is known to berelatively stable in-vivo. See Al-Obeidi et al. J. Amer. Chem. Soc.111:3413 (1989); Haskell-Luevano et al. J. Med. Chem. 39:432 (1996). Theunderlined portion indicates those residues within the cyclized portionof the peptide, and also the termini of the cyclic structure, i.e. thepeptide is cyclized by an amide bond from the side chains of asparticacid and lysine. This cyclic structure is used as a basis forconstructing labeled peptides according to the present invention.

Cyclic chelating derivatives based upon the structure of the known α-MSHreceptor binding ligand include those with a chelating derivativeattached to the N-terminus of the peptide, either directly or via aspacer group, such as γ-amino butyric acid (γ-Abu). Specific chelatingderivatives of this general structure include, but are not limited to:

-   MaGCγAbuNleDHF _(d) RWK-NH₂ (SEQ ID NO:1)-   PtscGCNleDHF _(d) RWK-NH₂ (SEQ ID NO:30)-   AcCGCNleDHF _(d) RWK-NH₂ (SEQ ID NO:31)-   DTPA-NleDHF _(d) RWK-NH₂ (SEQ ID NO:1)-   where-   Ma is mercaptoacetic acid,-   γAbu is γ-aminobutyric acid,-   PtscG is 2-(4-phenyl-3-thiosemicarbazidyl)acetic acid, and-   DTPA is diethylenetriaminepentaacetic acid.

In each of the compounds described above, the chelating moiety may bereplaced by a chelator of the general formula I, as described above.

(iii) Cyclic VIP R ceptor Targeting Agents

Naturally occurring VIP has the sequence:

-   HSDAVFTDNYTRLRKQMAVKKYLNSILN-NH₂ (SEQ ID NO:2)

Native VIP is thought to form a helical structure in solution. See Mussoet al. Biochemistry 27:8174 (1988). The putative helix structure can bestabilized by intramolecular cyclization via the side chains of residuesplaced in spatial proximity by the helical structure. Examples include:

-   -   Ac-HSDAVFTENYTKLRKQNleAAKKYLNDLKKGGT-NH₂ (SEQ ID NO:10)    -   Ac-HSDAVFTDNYTKLRKQNleAVKKYLNSVLT-NH₂ (SEQ ID NO:32)        (where Nle is norleucine). See O'Donnell et al. J. Pharm. Exp.        Ther. 270:1282; U.S. Pat. No. 4,822,890; Bolin, Eur. Pat. Appl.        0 536 741 A2. The underlined portion indicates the residues        within the cyclized portion of the peptide, and also the termini        of the cyclized portion, i.e. the peptide is cyclized via the        formation of an amide bond between the side chains of the        aspartic acid and the lysine. These cyclic structures are used        as a basis for constructing labeled peptides according to the        present invention.

Cyclic chelating derivatives based on these structures include, but arenot limited to, those with a metal binding moiety attached, eitherdirectly or via a spacer group, to the pharmacophore via the side chainamine of a lysine or other bis-amino acid residue. Specific chelatingderivatives of this general structure include, but are not limited to:

-   -   Ac-HSDAVFTENYTKLRK(PtscGC)QNleAAKKYLNDLKKGGT-NH₂ (SEQ ID NO:10)        (where PtscG=2-(4-phenyl-3-thioseinicarbazidyl)acetic acid); and    -   Ac-HSDAVFTENYTKLRK(DPTA)QNleAAKKYLNDLKKGGT-NH₂ (SEQ ID NO:10)        (where DTPA=diethylenetriaminepentaacetic acid).

In each of the compounds described above, the chelating moiety may bereplaced by a chelator of the general formula I, as described above.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLES Example 1 Synthesis of N^(α)Alloc-N^(ε)-Fmoc-L-Lysine

N^(ε)-Fmoc-L-Lysine (10.00 g, 27.1 mmol, 100 mol %, Bachem Biosciences,Inc.) was suspended in dioxane (100 ml) and Na₂CO₃ (1M, 33 ml) to form amilky suspension. Allyl chloroformate (3.2 ml, 30.2 mmol, 111 mol %) wasadded to dioxane (10 ml) and this solution was added dropwise to thesuspension of N^(ε)-Fmoc-L-Lysine over 10 min. Sodium carbonate, (1M, 20ml) was added in two portions and an additional quantity of allylchloroformate (0.3 ml) was added. The reaction was stirred at roomtemperature for 16 hours. The volatile solvents were removed underreduced pressure and the residue was washed with diethyl ether (50 ml).The residual liquid was then acidified with HCl (1M) and extracted withethyl acetate (2×150 ml). The organic layers were combined, washed withsaturated NaCl (50 ml), dried over Na₂SO₄, evaporated under reducedpressure to obtain a crude oily product (16 g). The crude product wasdissolved in ether (100 ml) and a white solid formed and was removed byfiltration. The solvent from the filtrate was removed under reducedpressure to afford a viscous pale yellow oil (8.34 g, 68% yield) whicheventually formed a glassy solid.

Example 2 Synthesis of 2-(triphenylmethylmercapto) acetyl hydrazide

2-(triphenylmethylmercapto) acetic acid (20.35° g, 60.9 mmol, 100 mol %)was dissolved in anhydrous THF (150 ml) and cooled in an ice water bath.t-Butylcarbazate (8.61 g, 65.1 mmol, 107 mol %) was added to thereaction solution followed by diisopropylcarbodiimide (10.0 ml, 63.9mmol, 105 mol %). The reaction was allowed to warm slowly to roomtemperature and stirred for 28 hours. The reaction mixture was filteredto remove the white precipitate that had formed and the filtrate wasconcentrated to a white foam by removal of the solvent under reducedpressure. This material was dissolved in chloroform (75 ml). Then aceticacid (75 ml) was added followed by the addition of borontrifluorideetherate (10.0 ml, 81 mmol, 134 mol %). The reaction was stirred at roomtemperature for 6 hours and then quenched by pouring the reactionmixture into water (200 ml) containing sodium acetate (30 g). Thismixture was extracted with chloroform (2×100 ml). The organic layerswere combined, washed with saturated NaCl solution (150 ml), dried overNa₂SO₄ and filtered. The solvent was removed under reduced pressure toobtain a pale gold oil which solidified on standing. The solid wassuspended in 1:1 diethylether/hexanes (200 ml) and collected byfiltration. The solid was washed with an additional quantity of 1:1diethylether/hexanes (100 ml) and dried to afford the desired product(15.44 g, 73% yield) having ESMS MH⁺ calculated 349, observed 349.

Example 3 Synthesis of N^(β)-[2-(triphenylmethylthio) acetyl]azaglycine

Glyoxylic acid monohydrate (0.59 g, 6.41 mmol, 110 mol %) was dissolvedin methanol (20 ml) and 2-(triphenylmethylmercapto)acetyl hydrazide(2.03 g, 5.82 mmol, 100 mol %) was added. Dioxane (20 ml) was added tothe cloudy reaction mixture and the reaction was stirred at roomtemperature for 18 hours. Sodium borohydride (1.76 g) was added to thereaction mixture and after 30 minutes, another quantity of sodiumborohydride (0.60 g) was added. The reaction was stirred for 3 hours atroom temperature, then quenched by pouring the reaction mixture into HCl(1M, 60 ml). The mixture was extracted with ethyl acetate (2×50 ml). Theorganic layers were combined, washed with saturated NaCl solution (40ml), dried over Na₂SO₄, filtered, and concentrated under reducedpressure on the rotary evaporator to afford a solid (2.5 g) having ESMSMH⁺ calculated 407, found 407.

Example 4 Synthesis of N^(α)-Boc-N^(β)-[2-(triphenylmethylthio)acetyl]azaglycine

N^(β)-[2-(triphenylmethylthio)acetyl]azaglycine (2.39 g, 5.89 mmol, 100mol %) was dissolved in dioxane (50 ml). Di-t-butyl dicarbonate (BOC)₂O,(2.07 g, 9.48 mmol, 161 mol %) was added to the reaction solutionfollowed by the addition of Na₂CO₃ (1M, 15 ml). This mixture was stirredat room temperature for 15 minutes, then additional quantities of Na₂CO₃(1M, 10 ml) and (BOC)₂O (1.41 g) were added. The solution was stirred atroom temperature for 18 hours then reacted with NaOH (6M, 3 ml) and(BOC)₂O (1.4 g) for 1 hour. The crude reaction mixture was thenacidified to pH 3 with citric acid (1M) and extracted with ethyl acetate(200 ml). The organic layer was washed with saturated sodium chloridesolution (60 ml), dried over Na₂SO₄, filtered and concentrated underreduced pressure to obtain the crude product. The crude product wasdissolved in ether and diluted to obtain a 1:1 mixture with hexanescausing a white precipitate to form. The white solid was collected byfiltration to obtain the desired product (1.48 g, 50% yield) having ESMSMH⁺ calculated 507, found 507.

Example 5 Synthesis of 2-(4-Phenyl-3thiosemicarbazidyl)acetic acid

4-Phenyl-3-thiosemicarbazide (6.02 g, 36 mmol, 100 μmol %) was suspendedin methanol (40 ml). Glyoxylic acid monohydrate (3.32 g, 36.1 mmol, 100mol %) was added and the reaction was stirred at room temperature for 2hours. Sodium borohydride (1.50 g) was added carefully, and the reactionmixture bubbled-very vigorously. The reaction mixture was stirred atroom temperature for 1 hour, then NaBH₄ (0.66 g) was added, followed bythe addition of glacial acetic acid (6 ml). After 15 minutes, NaBH₄(1.08 g) was added, and the reaction was stirred at room temperature for15 hours. An additional quantity of NaBH₄ (1.66 g) was then added andthe reaction was stirred at room temperature for 3 hours, before it wasquenched with HCl (1M, 200 ml). The mixture was then extracted withethyl acetate (2×150 ml). The organic layers were combined, washed withsaturated NaCl solution (100 ml), dried over Na₂SO₄, filtered, and thesolvent removed under reduced pressure to afford a yellow solid (9.03 g)having ESMS Negative ion mode M-H⁺ Calculated 224 Found 224.

Example 6 Synthesis of N^(β)-Boc-2-(4-Phenyl-3-thiosemicarbazidyl)aceticacid

2-(4-Phenyl-3-thiosemicarbazidyl)acetic acid (8.93 g, 37.9 mmol, 100 mol%) and (BOC)₂O (9.10° g) were dissolved in dioxane (100 ml). Sodiumcarbonate (1M, 50 ml) and water (50 ml) were added and the mixture wasstirred at room temperature for 5 hours. Sodium hydroxide (1M, 40 ml)and an additional quantity of (BOC)₂O (6.21 g), were added and thereaction was stirred overnight at room temperature. The reaction wasquenched with citric acid (1M) and extracted with ethyl acetate (2×100ml). The organic layers were combined, washed with saturated NaCl (50ml), dried over Na₂SO₄, and filtered. The filtrate was concentratedunder reduced pressure to afford a gummy solid (19 g). The crude solidwas suspended in ether and a white solid was collected by filtration.The solid was washed with ether (100 ml) to obtain the desired product(3.17 g) having ESMS MH⁺ calculated 326, found 326.

Example 7 Synthesis ofN^(α)-(triphenylmethylsulfenyl)-N^(β)-(Boc)azaglycine

^(t)-Butylcarbazate was condensed with glyoxylic acid monohydrate inmethanol. This crude hydrazone was then reduced by catalytichydrogenation over 10% Pd/C. This product was then mixed with dioxaneand base and a dioxane solution of triphenylmethanesulfenylchloride wasadded dropwise. The desired N^(α)-(triphenylmethylsulfenyl)-N^(β)-(Boc)azaglycine (25 g) was obtained on work-up.

Example 8 Solid Phase Peptide Synthesis of Peptides Using Alloc and FmocProtecting Groups

Solid phase peptide synthesis was carried out on a 0.050 mmol scaleusing an Advanced ChemTech model 348 peptide synthesizer modified tooperate under nitrogen pressure in the same manner as the model 396. Theallyloxycarbonyl (aloc) and 9-fluorenylmethyloxycarbonyl (Fmoc) groupswere employed for nitrogen protection and diisopropylcarbodiimide(DIC)/hydroxybenzotriazole (HOBT) were used to activate the carboxylgroups for coupling. A variety of resins were used such as Rink, Pal,and TentaGel S RAM for C-terminal amides and Wang, 2-chlorotrityl, orTentaGel S PHB for C-terminal acids.

To allow either introduction of the metal binding chelate moiety and/orto allow cyclization via selectively deprotected amino acid side chainsa differentially protected bis-amino acid was used for the peptidesynthesis. The differentially protected bis-amino acid derivativeschosen were α-Aloc-Lys(ε-Fmoc)OH and α-Fmoc-Lys(ε-Aloc)OH. Theα-Aloc-Lys(ε-Fmoc)OH derivative allowed the ligand pieces to beintroduced on the side chain using a routine Fmoc procedure.

The aloc groups were cleaved on the machine in the manual mode bywashing the resin bound peptide with dichloromethane (3×2 ml portions)and then mixing the resin with a solution (2 ml) containingtetrakistriphenylphosphine palladium [0] (10 mg), and acetic acid (0.1ml). Tributyltinhydride (0.3 ml) was then added and the mixture wasvortexed for one hour. The reaction cell was then emptied, the resin waswashed with dichloromethane (3×2 ml) and standard Fmoc synthesis wasthen resumed. The peptides were cleaved from the resin with a solutionof trifluoroacetic acid (TFA), anisole and ethane dithiol for 1 to 3hours in the ratio 23:3:1. The crude cleavage mixture was then pouredinto ether to precipitate the crude peptide which was then purified byreverse phase HPLC using a Waters Delta. Pak, Prep Pak C-18 cartridgesystem eluted with an appropriate gradient of TFA (0.1%) in water and/orTFA (0.1%) in acetonitrile (90%) and water (10%). The fractionscontaining the desired purified peptides were collected and the volatilesolvents were removed under reduced pressure to obtain the aqueoussolutions of the peptides which were then lyophilized. Samples of thelyophilized products were then sent for electrospray (ESMS) or fast atombombardment (FABMS) to confirm that the observed mass of the productsmatched the calculated mass of the desired peptide.

The table below shows some of the peptide sequences synthesized by themethods described above.

Peptide HPLC^(a) MW^(b) <GHWSYGLRPG—NH₂ (SEQ ID NO:24) 6.1 1183<GHYSLEWKPG—NH₂ (SEQ ID NO:33) 6.2 1227 HSDAVFTDNYTRLRKQMAVKKYLNSILN—NH₂(SEQ ID NO:2) 6.7 3326 MaGCγAbuHSDAVFTDNYTRLRKQMAVKKYLNSILN—NH₂ (SEQ IDNO:2) 7.3 3645 MaGCγAbUVFTDNYTRLRKQMAVKKYLNSILN—NH₂ (SEQ ID NO:4) 7.53235 MaGCγAbuNleDHFR_(d) WK—NH2^(c) (SEQ ID NO:1) 7.0 1302<GHWSYK(MaGC)LRPG—NH₂ (SEQ ID NO:6) 6.3 1488 <GHYSLK(MaGC)WKPG—NH₂ (SEQID NO:7) 6.3 1460 <GHWSYK(Ma-azaGC)LRPG—NH₂ (SEQ ID NO:6) 6.1 1503<GHYSLK(PtscGC)WKPG—NH₂ (SEQ ID NO:7) 6.9 1536 AcNal_(d) Cpa_(d) W_(d)SRK_(d) (MaGC)LRPA_(d) —NH₂ (SEQ ID NO:8) 8.2 1668<GHYSYLK(PtscGDap)WKPG—NH₂ (SEQ ID NO:11) 6.6 1519<GHYSLK(azaGGC)WKPG—NH₂ (SEQ ID NO:7) 6.5 1474 Nal_(d) Cpa_(d) W_(d)SRK_(d) (PtscGC)WKPG—NH₂ (SEQ ID NO:12) 8.1 1701 <GHWSYK_(d)(MaGC)LRPG—NH₂ (SEQ ID NO:13) 6.3 1488 AcNal_(d) Cpa_(d) W_(d) SRK_(d)(AzaGFC)LRPA_(d) —NH₂ (SEQ ID NO:8) AcNal_(d) Cpa_(d) W_(d) SRK_(d)(MaFC)LRPA_(d) —NH₂ (SEQ ID NO:8) AcNal_(d) Cpa_(d) W_(d) SRK_(d)(PtscGC)LRPA_(d) —NH₂ (SEQ ID NO:8) <GHWSYK(iDGDap)LRPG—NH₂ (SEQ IDNO:6) <GHWSYK(iECG)LRPG—NH₂ (SEQ ID NO:9) ^(a)HPLC Method [retentiontime in minutes] Solvent A is 0.1% trifluoroacetic acid in water,Solvent B is 0.1% trifluoroacetic acid in 90:10 acetonitrile/water.Solvent flow rate is 3 ml/min for 10 min, then 5 ml/min for 5 min.Gradient is 0 to 100% B over 10 min then 100% B for 5 min^(b)Electrospray mass spectrum values (MH⁺) ^(c)The underlined sequenceis cyclized as the cyclic amide connecting the side chain functionalgroups Abbreviations used in Table: <G: pyroglutamic acid PtscG:2-(4-phenyl-3-thiosemicarbazidyl)acetic acid or PhNHCSNHNHCH₂CO₂H Ma:mercaptoacetic acid azaG: azaglycine or H₂NNHCH₂CO₂H Dap:2,3-diaminopropionic acid γAbu: γ-aminobutyric acid Nal:2-naphthylalanine Cpa: 4-chlorophenylalanine K_(d): the subscript ddenotes that the D isomer was used K(MaGC): the parentheses denote thatenclosed amino acids are attached to the ε amine of lysine and the firstamino acid attached is C followed by G and ending in Ma iD: isoasparticacid iE: isoglutamic acid

Sequence MH+ HPLC RT AcK(TscGC)F_(d) CFW _(d) KTCT-OH (SEQ ID NO:14)1436 7.7 AcK(TscGC)DF_(d) CFW _(d) KTCT-OH (SEQ ID NO:15) 1552 7.4TscGCDF_(d) CFW _(d) KTCT-OH (SEQ ID NO:34) 1381 7.7 AcK(TscGC)F_(d) CFW_(d) KTCT-ol (SEQ ID NO:14) 1422 7.6 AcK(MtscGC)F_(d) CFW _(d) KTCT-ol(SEQ ID NO:14) 1436 7.8 AcK(TscGC)DF_(d) CFW _(d) KTCT-ol (SEQ ID NO:15)1537 7.4 AcK(MaGG)F_(d) CFW _(d) KTCT-ol (SEQ ID NO:14) 1378 7.4K(TscGC)DF_(d) CFW _(d) KTCT-NH₂ (SEQ ID NO:15) 1508 7.1 K(TscGC)KKF_(d)CFW _(d) KTCT-ol (SEQ ID NO:17) 1651 7.2 K(TscGC)KDF_(d) CFW _(d)KTCT-OH (SEQ ID NO:18) 1637 7.3 K(TscGC)DF_(d) CFW _(d) KTCT-ol (SEQ IDNO:15) 1495 7.2 K(TscGC)DSF_(d) CFW _(d) KTCT-OH (SEQ ID NO:19) 1596 7.4K(TscGC)DF_(d) CFW _(d) KTCT-OH (SEQ ID NO:15) 1508 7.2 K(TscGC)DF_(d)CFW _(d) KTCD-NH₂ (SEQ ID NO:20) 1521 7.1 K(TscGC)KDF_(d) CFW _(d)KTCT-NHNH (SEQ ID NO:18) 1651 7.2 AcK(TscGC)F_(d) CFW _(d) KTCT-NHNH₂(SEQ ID NO:14) 1450 7.4 K(AGC)F_(d) CFW _(d) KTCT-ol (SEQ ID NO:14) 13796.8 AcK(TscGC)DF_(d) CFW _(d) KTCT-ol (SEQ ID NO:15) 1537 7.4 F_(d) CFW_(d) KTCTK(TscGC)-NH₂ (SEQ ID NO:21) 1393 6.8The underlined portion of the sequence is cyclic.

-   TscG is 3-thiosemicarbazonylglyoxyl, i.e. H₂NCSNHNCHCO—-   MtscG is 4-methyl-3-thiosemicarbazonylglyoxyl,-   i.e. CH₃NHCSNHCHCO—-   Ma is mercaptoacetyl: HSCH₂CO—-   Groups listed within parentheses are attached to the side chain of    the amino acid to the left of the parentheses

Example 9 Preparation of a Cyclic MSH Analogue Containing a ChelatingMoiety

The method of synthesizing cyclic peptides was demonstrated by preparingthe cyclic α-melanocyte stimulating hormone (αMSH) analogueMaGCγ-AbuNleDHF _(d) RWK-NH₂, (SEQ ID NO:1) where the underliningindicates that the peptide sequence is cyclized as a lactam through theaspartic acid and lysine side chains. The residues to be used forcyclization were side-chain protected as the aloc group (for lysine) andas the allyl ester (for aspartate). The peptide was assembled using Fmocchemistry as described above, on a polystyrene-based Rink amide resin.

Allyl and aloc deprotection was first carried out using Pd(PPh₃)₄,acetic acid, and Bu₃SnH in the absence of piperidine as an allylscavenger. After cleavage of the side chain protecting groups, the resinwas washed and the partially protected peptide was cyclized using themethod described by Felix et al., Int. J. Peptide Protein Res. 32:441(1988). The peptide was then cleaved from the resin and purified toisolate the N-allyl substituted cyclic amide as the only clean peptidefrom the product mixture.

The aloc cleavage reaction was then modified by the addition ofpiperidine as an allyl scavenger.

When the aloc and allyl groups were cleaved using a mixture containing0.5 ml glacial acetic acid, 10 ml dichloromethane, 0.0563 gtetrakis(triphenylphosphine)palladium (0), plus 1.0 ml piperidine as anallyl scavenger. Each well on the peptide synthesizer contained 0.05mmol of peptide on Rink resin, and was treated with 0.3 ml/welltributyltin hydride at room temperature for 1 hr with vortex mixing. Theresin was washed with: 2×2 ml/well dichloromethane, 2×1 ml/wellmethanol, 2×1 ml/well diisopropylethyl amine, and 3×1 ml/well NMP afterthe cleavage of the side chain protecting groups. The peptide sidechains were then coupled by the method of Felix supra (15 hr, using BOPand DIEA). The peptide was cleaved and purified as described above toafford a pure peptide with the desired ESMS MH⁺ of 1302. The N-allylatedside product was observed in only trace amounts.

Example 10 Radiolabeling with ^(99m)Tc

A Glucoscan (DuPont) vial was reconstituted with 2.18 mCi of NaTcO₄ in 1ml saline to form the ^(99m)Tc-gluceptate complex. <GHWSYK(MaGC)LRPGamide (SEQ ID NO:6) (IMP3) was prepared as above 99m Tc-IMP₃ wasprepared by mixing 360 μl (874 uCi) of ^(99m)Tc-gluceptate with 640 μlof peptide in saline. The initially formed precipitate disappeared uponheating for 15 min at 75.degree°. An instant TLC (ITLC) strip developedin H₂O:EtOH:NH₄OH mixture (5:2:1) showed 6.2% of the activity at theorigin as colloids. HPLC showed 100% of the activity bound to thepeptide with a RT of 6.95 min, whereas the unlabeled peptide eluted at6.4 min under the same HPLC conditions (reversed phase C-18 column,gradient of 0–100% B in 10 min at a flow rate of 3 ml/min, where A is0.1% TFA in H₂O and B is 90% CH₃ CN, 0.1% TFA). Recovery from the HPLCcolumn was 85% of the injected activity.

IMP3 was formulated and lyophilized for ^(99m)Tc labeling in the amountsshown below:

IMP3 (μg) Sn (μg) αDG/SN 1. 250 23 14 2. 100 23 14 3. 250 15 14where αDG is α-D-glucoheptonate. The lyophilized vials werereconstituted with ˜900 μuCi of NaTcO₄ in saline. Cloudiness wasobserved in all the vials. The vials were heated for 15 min at 75° C.,but turbidity persisted. ITLC analysis for colloids showed 14, 21 and 9%colloids at the origin for vials 1, 2, and 3, respectively.

In order to prevent the precipitation during ^(99m)Tc labeling,α-D-glucoheptonate (αDG) and tartrate ratios to Sn(II) were varied inthe lyophilized vials. The following vials were formulated andlyophilized (250 μg of IMP3 with 25 μg Sn(II)) with tartrate and αDGratios as shown below. The vials were reconstituted with ˜500 μCi ofNaTcO4 in 1 ml saline. Observations are indicated in the observationcolumn. ITLC strips were developed after 15 min at room temperaturefollowing heating at 75° C. for 15 min.

tartrate/Sn pH Observation colloid, RTcolloid, 75° C. 1. 50 5.3 ppt 2.100 5.3 ppt 3. 500 5.3 ppt clears 17% 2.4% upon mixing αDG/Sn pHObservation colloid, RTcolloid, 75° C. 4. 25 5.3 ppt 5. 50 5.3 ppt 6.100 5.3 turbid 7. 500 5.3 slight turbidity 25% 3.5% 8. 1000 5.3 clear3.3% 3.1% The protocol above was repeated for vials 3, 7 and 8 and colloids weredetermined to be 5.3, 3.8, and 4.6%, respectively after heating 15 minat 75° C. A single broad peak was observed on a reversed HPLC column ata RT of 7 min.

Solubility of peptides that are poorly soluble in saline alone isincreased by the addition of a solubilizing agent such as ethanol or2-hydroxypropyl-βcyclodextrin.

Results from labeling other peptides with technetium-99 are shown in thetable below:

HPLC HPLC retention retention Peptide time^(a) time^(b)MaGCγAbuHSDAVFTDNYTRLRKQMAVKKYLNSILN—NH₂ (SEQ ID NO:2) 7.62 (99%) 7.65MaGCγAbuVFTDNYTRLRKQMAVKKYLNSILN—NH₂ (SEQ ID NO:4) 7.8–9.7^(e) 8.19^(c)(99%) <GHWSYK(MaGC)LRPG.amide (SEQ ID NO:6) 6.59 (95%) 6.90^(c) (92%)<GHYSLK(MaGC)WKPG.amide (SEQ ID NO:7) NA 7.07 (100%)<GHWSYK(Ma-azaGC)LRPG.amide (SEQ ID NO:6) 6.82 (100%) 7.02^(c) (99%)<GHYSLK(Ptsc-GC)WKPG.amide (SEQ ID NO:7) 7.60 (100%) 7.67^(d) (100%)AcNal_(d) Cpa_(d) W_(d) SRK_(d) (MaGC)LRPA_(d) —NH₂ (SEQ ID NO:8) 8.50(27%) 9.00 (68%) <GHWSYK_(d) (MaGC)LRPG—NH₂ (SEQ ID NO:13) 6.83 (95%)7.07^(c) (95%) <GHYSYLK(PtscGDap)WKPG—NH₂ (SEQ ID NO:11) 7.08 (96%)6–8^(e) (90%) <GHYSLK(azaGGC)WKPG—NH₂ (SEQ ID NO:7) 6.60 (100%) 6.47^(c)(99%) Nal_(d) Cpa_(d) W_(d) SRK_(d) (PtscGC)WKPG—NH_(d) (SEQ ID NO:12)8.43 (97%) Abbreviations used in the table are the same as in Example 8supra. A change in HPLC retention time of the complex formed by labelingat room temperature and that formed by heating indicates a change in thebinding of the metal. ^(a)room temperature reaction 15 min [retentiontime in minutes] ^(b)after heating in boiling water 15 min^(c)significant change in peak shape and retention after heating ^(d)nochange in peak shape and retention time difference is not significant^(e)many peaks

In an alternative labeling method, dilute solutions (30 μg/mL) of thepeptide were formulated into labeling kits prior to addition ofpertechnetate. The final solution contained the peptide, 10%hydroxypropyl-βcyclodextrin (HPCD), 200 mM glucoheptonate 21 mM acetatebuffer at pH 5.3, 2 mg of ascorbic acid and 100 μg of stannous chloridein 1.5 mL total volume. In other formulations, two equivalents ofstannous ion relative to the peptide were added, in a buffer containing15% HPCD, 200 mM glucoheptonate, and 21 mM acetate buffer at pH 5.6.

Example 11 Radiolabeling of IMP-3 ¹⁸⁸Re

IMP3, (<GHWSYK(MaGC)LRPG amide) (SEQ ID NO:6) was synthesized as above.IMP3 has a retention time of 6.4 min on a reversed phase C-18 columnusing a gradient of 0–100% B in 10 min at a flow rate of 3 ml/min whereA is 0.1% TFA in H₂O and B is 90% CH₃ CN, 0.1% TFA.

IMP3 was formulated in 1 mg and 250 μg amounts with 450 μg Sn(II) andα-D-glucoheptonate at a ratio of 1:17.5, and lyophilized. Thelyophilized vials of IMP3 (1 mg and 250 μg) were reconstituted with 617and 578 μCi of NaReO₄ in saline. The vials were heated for 15 min at 75°C. HPLC analysis under the conditions described above showed singlepeaks at RT of 7.0 min for both vials. The effluent was collected andcounted on a γ-counter. For the 1 mg vial, the recovery of activity was88% whereas the recovery was 77% for the 250 μg vial. Colloid analyseson an ITLC strip developed in H₂O:EtOH:NH₄OH(5:2:1) showed 1.4 and 1.2%of the activity at the origin for 1 mg and 250 μg vials, respectively.

¹⁸⁸Re labeling at room temperature did not proceed as well as at 75° C.At room temperature, only a few percent of the activity (<5%) wasincorporated into the peptide and the rest of the activity eluted in thevoid volume (1.2 min).

Example 12 In vitro Receptor Binding Assays

The human breast adenocarcinoma cell lines MCF-7, SK-BR-3, andMDA-MB-231 were used for testing radiometal labeled LHRH analogues. HT29cells were used for testing labeled VIP analogues. All cells lines werepurchased from the American Type Culture Collection, Rockville, Md.Cells were grown in DMEM supplemented with 5% fetal bovine serum, 5%defined equine serum, penicillin (100 U/ml), streptomycin (100 μg/ml),and L-glutamine (2 mM). The cells were routinely passaged afterdetachment with trypsin and 0.2% EDTA.

Specificity of the unlabeled LHRH analogue peptides is determined bycompetitive cell binding assay. Target cells are washed with freshmedium, and adjusted to 5×10⁵ cell/ml. 100 μl of the cell suspension(100 μl) is added per well to a 96-well microtiter plate. The cells areallowed to attach and are then treated with different concentrations ofthe peptides in the presence of ¹²⁵I-LHRH (Amersham Life Science,Arlington Heights, Ill., 2,000 Ci/mmol). Following a 2 h incubation atroom temperature with shaking, the cells are washed twice and theradioactivity associated with the cells is counted and the concentrationof the peptides that cause 50% inhibition on the binding of the labeledLH-RH is compared.

To determine receptor binding constants, serial dilutions ofradiolabeled LHRH are incubated with 5×10⁵ cells in a 96-well plate. Allassay are performed in triplicates both with or without a highconcentration of unlabeled LHRH to allow determination of specificallybound peptide. After a 2 h incubation at room temperature, the cells arewashed and counted. The equilibrium association constant, K_(a), and thetotal number of receptor sites per cell are determined by Scatchardanalysis.

For testing VIP analogues and their metal complexes the protocoldescribed Virgolini et al. (Cancer Res. 54:690 (1994)) is used. Briefly,¹²⁵I VIP is mixed with increasing concentrations of test peptide in asolution of binding buffer, following which each solution is added toHT29 cells in a 48 well culture plate. Each concentration is tested intriplicate. The cells are incubated at 4° C. for 2 h, followed by threewashes with ice-cold binding buffer. The cells are then lysed with 2MNaOH for 5 min and the liquid in the well is removed with a cotton swab.The activity on the cotton swab is counted using a gamma counter.

The invention has been disclosed broadly and illustrated in reference torepresentative embodiments described above. Those skilled in the artwill recognize that various modifications can be made to the presentinvention without departing from the spirit and scope thereof. Subjectmatter relating to radiometal-binding peptides also is described incopending U.S. application Ser. No. 08/474,555, the disclosure of whichis hereby incorporated by reference in its entirety.

1. A method of treating a tumor, comprising administering to a humanpatient a radiolabeled peptide and a pharmaceutically acceptablecarrier, wherein said peptide comprises a radiometal-binding moietycomprising the structure:

wherein R¹, R², and R³ independently are selected from the groupconsisting of H, C₁–C₆ alkyl, substituted C₁–C₆ alkyl, C₃–C₆ cycloalkyl,substituted C₃–C₆ cycloalkyl, heterocycloalkyl, C₆–C₁₂ aryl, C₆–C₁₂substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, and aprotecting group, provided that at least one of R¹, R², or R³ is H, R⁵,R⁷, R⁸, R⁹ and R¹⁰ independently are selected from the group consistingof H, C₁–C₆ alkyl, substituted C₁–C₆ alkyl, C₆–C₁₂ aryl, and substitutedC₆–C₁₂ aryl, and R⁸ and R⁹ together or R⁷ and R⁹ together may form acycloalkyl or substituted cycloalkyl ring, R⁴ and R⁶ together form adirect bond or are independently selected from the group consisting ofC₁–C₆ alkyl, substituted C₁–C₆ alkyl, C₆–C₁₂ aryl, and substitutedC₆–C₁₂ aryl, and wherein NR¹⁰ is located at the N-terminus of saidpeptide, or is located on an amino acid side chain of said peptide.
 2. Amethod according to claim 1, wherein R¹ is H.
 3. A method according toclaim 1, wherein R³ is H.
 4. A method according to claim 1, wherein R⁴is H.
 5. A method according to claim 1, wherein R⁴ and R⁶ together forma direct bond.
 6. A method according to claim 1, wherein R⁵ is H.
 7. Amethod according to claim 1, wherein NR¹⁰ is located at the N-terminusof said peptide.
 8. A method according to claim 1, wherein NR¹⁰ islocated on an amino acid side chain of said peptide.
 9. A methodaccording to claim 2, wherein R² is lower alkyl or substituted orunsubstituted phenyl.
 10. A method according to claim 9, wherein R² isH.
 11. A method according to claim 10, wherein R³ is H.
 12. A methodaccording to claim 11, wherein R⁴ and R⁶ together form a direct bond.13. A method according to claim 11, wherein R⁵ is H.
 14. A methodaccording to claim 13, wherein R⁷, R⁸, and R⁹ each are H.
 15. A methodaccording to claim 14, wherein R² is phenyl.
 16. A method according toclaim 14, wherein R² is methyl.
 17. A method according to claim 1,wherein R⁸ and R⁹ are methyl.
 18. A method according to claim 1, whereinsaid peptide is selected from the group consisting of: (Chel)γAbuNleDHF_(d) RWK-NH₂, (SEQ ID NO:1) (Chel)γAbuHSDAVFTDNYTRLRKQMAVKKYLNSILN-NH₂,(SEQ ID NO:2) KPRRPYTDNYTRLRK(Chel)QMAVKKYLNSILN-NH₂, (SEQ ID NO:3)(Chel)γAbuVFTDNYTRLRKQMAVKKYLNSILN-NH₂,(Chel)γAbuYTRLRKQMAVKKYLNSILN-NH₂, (SEQ ID NO:4)HSDAVFTDNYTRLRK(Chel)QMAVKKYLNSILN-NH₂, (SEQ ID NO:5) (SEQ IDNO:6)<GHWSYK(Chel)LRPG-NH₂, <GHYSLK(Chel)WKPG-NH₂, (SEQ ID NO:7)AcNal_(d) Cpa_(d) W_(d) SRK_(d) (Chel)LRPA_(d)-NH₂, (SEQ ID NO:8) (SEQID NO:9) (Chel)AbuSYSNleDHF_(d) RWK-NH₂,Ac-HSDAVFTENYTKLRK(Chel)QNleAAKKYLNDLKKGGT-NH₂, (SEQ ID NO:10) (SEQ IDNO:12) Nal_(d) Cpa_(d) W_(d) SRK_(d) (Chel)WKPG-NH₂, <GHWSYK_(d)(Chel)LRPG-NH₂, (SEQ ID NO:13) (SEQ ID NO:14) AcK(Chel)F_(d) CFW _(d)KTCT-OH, AcK(Chel)DF_(d) CFW _(d) KTCT-OH, (SEQ ID NO:15) (SEQ ID NO:14)AcK(Chel)F_(d) CFW _(d) KTCT-ol, AcK(Chel)DF_(d) CFW _(d) KTCT-ol, (SEQID NO:15) (SEQ ID NO:16) (Chel)DF_(d) CFW _(d) KTCT-OH, K(Chel)DF_(d)CFW _(d) KTCT-ol, (SEQ ID NO:15) (SEQ ID NO:17) K(Chel)KKF_(d) CFW _(d)KTCT-ol, K(Chel)KDF_(d) CFW _(d) KTCT-OH, (SEQ ID NO:18) (SEQ ID NO:19)K(Chel)DSF_(d) CFW _(d) KTCT-OH, K(Chel)DF_(d) CFW _(d) KTCT-OH, (SEQ IDNO:15) (SEQ ID NO:20) K(Chel)DF_(d) CFW _(d) KTCD-NH₂, K(Chel)DF_(d) CFW_(d) KTCT-NH₂, (SEQ ID NO:15) (SEQ ID NO:18) K(Chel)KDF_(d) CFW _(d)KTCT-NHNH₂, AcK(Chel)F_(d) CFW _(d) KTCT-NHNH₂, (SEQ ID NO:14) (SEQ IDNO:14) K(Chel)F_(d) CFW _(d) KTCT-ol, and F_(d) CFW _(d)KTCTK(Chel)-NH₂, (SEQ ID NO:21) wherein (Chel) is a radiometal-bindingmoiety.
 19. A method according to claim 1, wherein said peptide containsat least one disulfide bond.
 20. A method according to claim 19, whereinsaid peptide is a polypeptide.